Estudio del efecto de dasatinib en combinación con cetuximab en un modelo animal de radioterapia experimental

La hiperactividad del EGFR (epidermal growth factor receptor) confiere mal pronóstico. El EGFR se encuentra sobreexpresado en distintos tipos de tumores, entre ellos el cáncer de colon, pulmón, páncreas y cabeza y cuello (HNSCC). Además la cooperación con otros miembros de la familia del EGFR podría ampliar esta lista a otras neoplasias. La inhibición del EGFR es una de las vías de investigación más activa contra el cáncer. La radioterapia consigue, en un elevado porcentaje de casos, una buena respuesta. Sin embargo, la existencia de células resistentes a la radiación reduce el control tumoral. Una de las causas de la radioresistencia es la hiperactividad del EGFR. En cáncer de cabeza y cuello, el bloqueo del EGFR con el anticuerpo monoclonal anti- EGFR cetuximab aumenta el control local y la supervivencia en pacientes tratados simultáneamente con radioterapia. Sin embargo, no todos los pacientes tratados con cetuximab y radiación se benefician del tratamiento, lo que sugiere que, independientemente de la expresión del EGFR, existen mecanismos de resistencia a la combinación. La ramificación de las vías de señalización que dependen del EGFR sugiere que la topografía de la resistencia puede ser variada y múltiple. Cuanto más arriba de la vía de transmisión de señales se sitúe el by-pass, mayor debería ser la resistencia debido a que un mayor número de funciones citoprotectoras se verían liberadas. Se han descrito distintas posibilidades de by-pass al bloqueo del EGFR. Una de las oncoproteínas que podría ejercer un by-pass proximal es SRC, cuya actividad se relaciona con la progresión tumoral tanto en fases avanzadas como en lesiones premalignas. Además, SRC y EGFR comparten vías de señalización que promueven la progresión de la célula tumoral. En conjunto estos hechos sugieren que la inhibición de SRC podría aumentar la potencia antitumoral de la combinación de radioterapia y cetuximab. En este proyecto se han evaluado los efectos del tratamiento concomitante con radioterapia, cetuximab y dasatinib, y mejorado los conocimientos de los mecanismos biológicos involucrados. También se ha determinado si el uso de inhibidores de SRC podía potenciar el efecto antitumoral de la combinación de radioterapia y cetuximab en tumores dependientes del EGFR. La adición de DST al cetuximab o al cetuximab y radioterapia tuvo un efecto contrario al esperado: produjo un mayor crecimiento de los tumores, un incremento de la angiogénesis y de la síntesis de ADN, un aumento de la fosforilación de las oncoproteínas RAS, ERK1/2, AKT y STAT3; una elevación de la secreción de VEGF, y una hiperactividad del metabolismo glucolítico. El tratamiento combinado con dasatinib e inhibidores de las quinasas JAK, HER2 y MET no inhibió el exceso de fosforilación de ERK inducido por dasatinib, una respuesta reactiva similar a la descrita con C225 y que sugiere la implicación de mecanismos de acción, comunes e independientes de JAK, HER2 y MET. Por el contrario el bloqueo de EGFR por el inhibidor tirosina quinasa AG1478 dio lugar a una inhibición completa de la fosforilación de ERK y AKT a pesar de la presencia de DST, indicando que la quinasa del EGFR está implicada en el efecto antagónico inducido por dasatinib. Por último, este trabajo contribuye a la idea que la combinación de terapias moleculares puede dar lugar a antagonismos, que pueden ponerse de manifiesto durante una investigación preclínica antes de ser evaluados en ensayos clínicos.Aberrant epidermal growth factor receptor (EGFR) signalling drives oncogenesis in many carcinomas, including head and neck squamous cell cancer (HNSCC). Irrespective of the mechanism of activation, EGFR overexpression is a major origin of resistance to cancer cell death—and a relevant cause of local failures after radiotherapy. The blockage of EGFR by the monoclonal antibody cetuximab in combination with radiotherapy has been shown to improve survival and local control in head and neck cancers. However, major responses are limited in part due to the robustness of signalling networking, which grants alternative routes to avoid blockage of EGFR. SRC-family kinases occupy a central position within the cell signal networking, which makes SRC a potential target in cancer treatment. In this project we evaluated the effects of the concomitant treatment with radiotherapy, cetuximab and dasatinib, and improved the knowledge of the biological mechanisms involved; to determine whether SRC inhibitors could potentiate the antitumor effects of radiotherapy and cetuximab combination in EGFR-dependent tumours. Unexpectedly, the addition of dasatinib to cetuximab or cetuximab and radiotherapy resulted in an antagonistic effect which was characterized by increased tumour growth; an increase in the angiogenesis and DNA synthesis; association with the inhibition of SRC; an increase in cell signalling involving phosphorylation of RAS, ERK1/2, AKT and STAT3 oncoproteins; elevation of VEGF secretion and hyperactivity of glycolytic metabolism. Also, the simultaneous blockade of SRC and JAK kinases, HER2 or MET, induced a similar reaction to that described with cetuximab suggesting the involvement of mechanisms of action, common and independent, of JAK, HER2 and MET. Conversely, blocking EGFR with the tyrosine kinase inhibitor AG1478 resulted in a complete inhibition of ERK and AKT phosphorylation, despite the presence of dasatinib, indicating that the EGFR kinase is involved in the induced antagonistic effect of dasatinib. Finally, this project contributes to the idea that the combination of molecular therapies can result in antagonism, which may become perceptible during a preclinical research before being evaluated in clinical trials

Simvastatin Enhances the Effects of Radiotherapy and Cetuximab on a Cell Line (FaDu) Derived from a Squamous Cell Carcinoma of Head and Neck

Radiotherapy (XRT) delivered with the antibody cetuximab is a standard treatment option for squamous cell carcinomas of head and neck (SCCNH). Cetuximab acts by blocking epidermal growth factor receptor (EGFR) signaling to inhibit cancer progression. However, a significant percentage of patients will not respond to XRT and cetuximab. Statins reduce the synthesis of cholesterol and isoprenoid derivates that may be required for efficient EGFR signaling. We assessed whether the statin simvastatin could improve this combined therapy. In vitro, simvastatin enhanced the effects of XRT alone and in combination with cetuximab in wound healing, cell proliferation, and clonogenic assays in FaDu cells. These results were reflected in xenoimplanted tumors growing into subcutaneous tissue of athymic mice where concomitant treatment with simvastatin decreased tumor growth. Consistently, lower levels of phosphorylated extracellular signal-regulated kinases 1 and 2, phosphatidylinositol 3-kinase/AKT-protein kinase B, and signal transducer and activator of transcription 3 oncoproteins and higher levels of caspase-3 and apoptosis in cell cultures and xenografts were observed. The EGFR-overexpressing A431 cell line was used to reproduce these antitumor effects of simvastatin. Our findings suggest that simvastatin may improve the efficiency of concomitant XRT and cetuximab. Further investigation in the treatment of SCCNH is warranted

The first hydrophobic region of the HPV16 E5 protein determines protein cellular location and facilitates anchorage-independent growth

The human papillomavirus type 16 E5 protein (HPV16 E5) is 83 amino acids in length and contains three well-defined hydrophobic regions. The protein is expressed at very limited amounts in transfected cells and the absence of specific antibodies has strongly hampered functional analyses. To investigate the relationship between structure and function we have synthesized a codon-adapted version of the gene (hE5) and prepared a series of N-terminal and C-terminal deletions. Immunofluorescence analyses show colocaliation of the protein with calnexin, an ER marker, EEA-1, an early endosomes marker, and Lamp-2, a lysosomal marker. No major colocalization was found between hE5 and the Golgi marker 58 K. Whereas deletions at the C-terminal end of the protein do not greatly alter the localisation pattern, deletion of the first hydrophobic region results in loss of colocalisation with the ER, early endosomes and lysosomes. Further, we show that while the complete E5 protein confers to HaCaT cells the property to grow in an anchorage-independent manner, deletion of the first hydrophobic region results in loss of growth in soft agar. We conclude that the first hydrophobic region of the E5 protein largely determines the biological properties of the viral protein

Guide of references for water-energy renovation and construction in the Sudoe area: characterisation of rural territories in the Sudoe area

The COLEOPTER (COncertation LocalE pour l’Optimisation des Politiques Territoriales pour l’Energie Rurale) project develops an integrated approach to the energy efficiency of public buildings that links technical, social and economic challenges. COLEOPTER addresses two energy efficiency challenges in buildings: difficulties for rural municipalities to act and carry out work despite the positive local impact (i.e., energy savings and local employment) and a lack of awareness of building challenges, which leads to irrational use of energy/water and low renovation rates.The COLEOPTER project (SOE3/P3/F0951) is financed by the Interreg Sudoe Programme that supports regional development in Southern Europe, financing transnational projects through the European Regional Development Fund.Preprin

Structural and Spectroscopic Behavior of double Metal Oxalates from the first transition Metal Series

A series of double metal oxalates of stoichiometry MM´(C2O4)2.4H2O (with MM´= MnCo, MnNi, MnZn, CoNi, CoCu, CoZn, NiCu, NiZn and CuZn have been prepared and characterized. Their structural behavior was investigated by means of X-ray powder diffractometry, showing a strong structural analogy to the orthorhombic, so-called beta-modification, of the related simple oxalato complexes of composition MC2O4.2H2O. The vibrational spectroscopic behavior of the double metal oxalates was investigated by infrared and Raman spectroscopy. The results clearly confirmed the close structural analogies between the MM´(C2O4)2.4H2O, alfa-MC2O4.2H2O and beta-MC2O4.2H2O series of complexes.Fil: D’Antonio, María C.. Universidad Nacional de la Patagonia Austral. Unidad Académica Río Gallegos; ArgentinaFil: Torres, María Marta. Universidad Nacional de la Patagonia Austral. Unidad Académica Río Gallegos; ArgentinaFil: Palacios, Daniel Alejandro. Universidad Nacional de la Patagonia Austral. Unidad Académica Río Gallegos; ArgentinaFil: Gonzalez Baro, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Química Inorgánica "Dr. Pedro J. Aymonino". Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Química Inorgánica "Dr. Pedro J. Aymonino"; ArgentinaFil: Barone, Vicente Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Química Inorgánica "Dr. Pedro J. Aymonino". Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Química Inorgánica "Dr. Pedro J. Aymonino"; ArgentinaFil: Baran, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Química Inorgánica "Dr. Pedro J. Aymonino". Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Química Inorgánica "Dr. Pedro J. Aymonino"; Argentin

Transgénesis en animales de granja

La transgénesis en animales de granja ha experimentado una importante evolución desde sus inicios en la década de los años 80. La eficiencia de las técnicas ha aumentado considerablemente y, a las primeras experiencias llevadas a cabo mediante microinyección pronuclear en el cigoto, le han seguido otras metodologías como la transferencia nuclear de células somáticas, que ha tenido particular importancia en estas especies domésticas. Además, en los últimos años, se han desarrollado herramientas de edición del genoma que permiten una alta especificidad en la modificación genética. Entre las numerosas aplicaciones de los animales transgénicos, se encuentra la producción de proteínas de uso terapéutico humano. También, cabe destacar las investigaciones con objeto de generar animales modificados genéticamente para ser utilizados como modelos de enfermedades humanas, o bien destinados a proporcionar órganos para xenotrasplante. Además, en el ámbito estricto de la Producción Animal se está explorando la utilización de la transgénesis para incrementar la resistencia de los animales a enfermedades o mejorar la cantidad y calidad de sus producto

The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

Background: Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. Methods: Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (gamma H2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. Results: We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by gamma H2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. Conclusions: The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay

The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

<p>Abstract</p> <p>Background</p> <p>Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair.</p> <p>Methods</p> <p>Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors.</p> <p>Results</p> <p>We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself.</p> <p>Conclusions</p> <p>The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay.</p